Abstract
Homodimeric H(+)-pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H(+)-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PP(i)), for H(+) translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PP(i) binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H(+)-PPase are 49.3 + or - 4.0 and 67.2 + or - 5.7 A, respectively. Furthermore, putative PP(i) binding motifs on individual subunits are found to be relatively far away from each other (70.8 + or - 4.8 A), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H(+)-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H(+)-PPase upon substrate binding.
Highlights
Hϩ-PPase3 is widely present in higher plants, as well as in some alga, protozoa, archaebacterial, and bacteria (1, 2)
The homodimeric mung bean Hϩ-PPase in the lipid bilayer had been visualized by atomic force microscopy to be larger in size at cytoplasmic protrusions but relatively smaller at luminal protrusions (12)
Construction and Characterization of CtHϩ-PPase Mutants—The DNA sequence and topological analyses suggest that the single polypeptide of CtHϩ-PPases is composed of 673 amino acids with its secondary structure containing 16 transmembrane domains (TMs), cytoplasmic and periplasmic loops, and
Summary
CtHϩ-PPase DNA Construction and Mutagenesis—The CtHϩPPase gene (GenBankTM accession number AA035020) was amplified from C. tetani E88 genomic DNA with Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) and cloned into pET23d vector (Novagen, Nottingham, UK) with a C-terminal His tag. The CtHϩ-PPase was solubilized from microsomal membrane (1 mg/ml) in an extraction buffer (25 mM MOPS/KOH (pH 7.2), 400 mM KCl, 4 mM MgCl2, 20% (w/v) glycerol, 1 mM phenylmethanesulfonyl fluoride, and 0.25% (w/v) n-dodecyl-D-maltopyranoside). Fluorescence Spectroscopy—Fluorophore-labeled CtHϩ-PPases (5 nM) were immobilized on the coverslips according to the previous protocol with minor modifications (12, 18). For single molecule imaging, purified CtHϩ-PPase in 10 mM MOPS/KOH (pH 7.2) and 0.05% (w/v) n-dodecyl--D-maltopyranoside was reacted with a 5-fold molar excess of both fluorophore Alexa Fluor 488 and 647, simultaneously. Distance calculation is based on the Forster theory with the energy transfer efficiency depending on the inverse sixth power of the distance between fluorescence donor and acceptor. A Forster distance of 56 Å between Alexa Fluor 488 and 647 was, obtained (25)
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